All isoprenoids are constructed by isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP). The essential role played by the deoxyxylulose phosphate (DXP) pathway in prokaryotic IPP and DMAPP biosynthesis and the lack of this pathway in animals makes the DXP pathway enzymes ideal candidates for developing broad-spectrum antibiotics. The DXP pathway is also targeted for herbicide development because its plant mutants are not able to synthesize sufficient amounts of carotenoids and chlorophylls for normal growth. The low natural abundance of isoprenoids has also stimulated interest in their production through bioengineering. Because the limiting factor in bioengineering-based isoprenoid production is the inadequate supply of IPP and DMAPP, mechanistic studies of the DXP pathway will guide the construction of host strains for bioengineering-based isoprenoid production. The proposed project will study the reaction mechanism of one of the DXP pathway rate-limiting steps, a reductive dehydration reaction catalyzed by an iron-sulfur cluster containing IspH protein. Several major achievements accomplished in preliminary studies serve as the basis for the proposed work. In the preliminary studies, IspH activity was improved by 97-fold relative to that reported in the literature. In addition, using substrate analogs, several IspH mechanistic options were examined and narrowed down to a model that is consistent with all current data. [57Fe]-labeled IspH was isolated in large quantities (~500 mg from a one-day purification). Initial EPR and Mvssbauer characterizations demonstrated that the IspH protein developed has both a high degree of iron- sulfur cluster load and homogeneity. Based on these achievements, the team has acquired all the necessary materials and protocols for conducting the proposed studies. Specifically: In Aim 1, both enzyme- and substrate-based intermediates will be trapped and characterized using a combination of bioorganic and biophysical methods. Several lines of evidence indicate that IspH exists as protein complexes. In Aim 2, by making use of the strains, reagents, and reporter systems obtained in the preliminary studies, several complementary approaches will be utilized to identify IspH partner proteins and study their functions.